SiRNA compositions and methods for treatment of HPV and other infections

ABSTRACT

The invention provides siRNA compositions that (1) interfere with viral replication of human papillomavirus (HPV), herpes simplex virus (HSV), and human immunodeficiency virus (HIV) in mucosal tissues, such as genital tissues, and (2) treat fungal infections. The compositions include siRNA molecules that target HPV, complexed with a dendrimer that treats and prevents genital herpes (HSV) and HIV. The compositions also include siRNA molecules that target HPV, complexed with a histidine-lysine (HK) polymer that treats and prevent fungus infection. The combined formulations of siRNA and dendrimer provide treatment of the infections from HPVs, HSVs, and HIVs. The combined formulations of siRNA and HK polymer provide treatment of HPVs and fungus infections.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation-in-part of, and claims the benefitunder 35 U.S.C. §§120 and 365(c) of, International Patent ApplicationNo. PCT/US2011/045884, filed Jul. 29, 2011, which claims priority to,and the benefit of, U.S. Provisional Patent Application No. 61/369,067,filed Jul. 29, 2010, the contents of each of which are expresslyincorporated by reference herein in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 4, 2013, isnamed SIR-009-P001-US_SL.txt and is 29,728 bytes in size.

FIELD OF INVENTION

The invention relates to siRNA molecules, compositions, and methods forthe treatment of human papillomavirus (HPV) infections and certain otherinfections, in particular, HIV, HSV, and fungal infections. Thecompositions include a cocktail of siRNA molecules targeted against HPV,formulated with dendrimer, a polymer which is effective against HIV andHSV infections. The compositions also include a cocktail of siRNAmolecules targeted against HPV, formulated with histidine-lysine (HK)polymer, a polymer which has been shown to be effective in vitro toinhibit fungal infections.

BACKGROUND HPV and Cervical Cancer

Human Papilloma Virus (HPV) is a group of coated DNA viruses, which nowhas over 100 different species, and is the most common sexuallytransmitted (ST) infection in adults worldwide. In 1976 Harald zurHausen from Germany published the hypothesis that human Papilloma virusplays an important role in the cause of cervical cancer tissue [1]. In1983 and 1984 zur Hausen and his collaborators identified HPV16 andHPV18 in cervical cancer [2-4]. Dr. zur Hausen was awarded the NobelPrize for Physiology and Medicine in 2008, because of his contribution.It is estimated that over 80% of US women by age 50 will have contractedat least one strain of HPV [5]. It is also estimated that each yearthere are 490,000 new cases of cervical cancer worldwide, which resultin 270,000 deaths. In the US, each year there are 250,000 to 1 millionwomen who develop cervical dysplasia, which leads to 11,000 furtherdeveloping cervical cancer, and to 4,000 deaths [6]. Among the 19“high-risk” HPVs which will lead to cervical cancer, HPV16 and 18 countfor about 70% of the cases [7].

HPVs have a circular genome of about 8 kb, with three major regions inthe genome, the early genes (E6, E7, E1, E2, E4 and E5), the late genes(L1 and L2), and the long control region (LCR) between L1 and E6. FIG. 1shows the characteristic HPV genome organization, using the medicallyimportant HPV-18 as the model. The early transcripts ending at 4215encode the 6 early genes, while the late transcripts ending at 7221encode the two late genes. E6 and E7 are cancer transforming proteinsbecause they inactivate tumor suppressor proteins p53 (inactivated byE6) and pRb (inactivated by E7) [8].

Although the US FDA has approved two HPV vaccines (below), there isstill a high demand for HPV therapeutics. However, there is no effectivetreatment on the market yet [9]. This invention describes HPVtherapeutics by siRNAs complexed with dendrimer and histidine-lysinepolymers.

HPV Vaccine

In 2006, the US FDA approved Gardasil®, an HPV vaccine produced byMerck, which is composed of hollow virus-like particles (VLP) assembledfrom recombinant HPV coat proteins and which targets HPV16, 18, 6 and11. The vaccine is aimed at use in women and girls. Later, it wasreported that Gardasil® is also effective in preventing genital warts inmales. The use of Gardasil® for men and boys was approved by the FDA onOct. 16, 2009. In October 2009, the FDA also approved Cervarix®, thesecond HPV vaccine targeting HPV16 and 18 and produced byGlaxoSmithKline [10].

Public health officials in industrialized counties and areas likeAustralia, Canada, Europe and the US recommend vaccination of youngwomen against HPV to prevent cervical cancer and genital warts, and toreduce the number of painful and costly treatments for cervicaldysplasia caused by HPV infections. It is recommended that women andgirls who are not exposed to HPVs between the ages of 9 to 25 should getan HPV vaccination [11]. However, many women and girls are notvaccinated because of various reasons. In the US, only about one-quarterof girls got HPV vaccination because most families worried about eitherthe effectiveness or the side effects of the vaccine [12]. In addition,HPV vaccines are not very easy to get access to in third worldcountries. In Kenya as an example, the cost of vaccination is over theaverage annual income of a family [13]. Furthermore, many women havebeen exposed to HPVs already [14].

Dendrimer and Clinical Trials for HIV and HSV Treatment and HPVInhibition In Vitro

Dendrimers are dendritic polymers which belong to a new class of polymerarchitecture (after traditional linear, cross-linked and branched types)[15]. VivaGel® (SPL7013), an anionic poly (L-Lysine) dendrimer, has beenapplied in at least 4 different clinical trials [16]. A phase I trial,completed on Jan. 20, 2008, tested male tolerance for topicalapplication of SPL7013. Phase I and II trials, completed on Jun. 4,2009, of vaginal dosing tested against HIV infection and HSV-2 herpes inwomen. A phase I trial, completed on Feb. 2, 2010, tested acceptabilityof SPL7013 in sexually active women. A phase I trial, completed on Mar.4, 2010, was a safety test for HSV-2 infection in health young women.Australian and US NCI laboratories also showed that SPL7013 inhibitsclinically relevant strains of HPV in vitro [17].

HK Polymers Inhibit Fungal Infections In Vitro

It was shown that histidine-lysine polymers are able to inhibit thegrowth of fungal strains, C. albicans and C. kefyr [18]. The highestinhibition activity was achieved by HK polymer H2K4b (MW, 11,137). Thestructure of H2K4b is as follows: KKK(KHKHHKHHKHHKHHKHHKHK)4 (coresequence disclosed as SEQ ID NO: 1)

RNAi and Development of Novel Therapeutics

RNA interference (RNAi) was first illustrated in plants, but quicklyproved to be a universal process covering low and high biologicalspecies. It is an efficient process in which double-stranded RNAduplexes were generated and lead to sequence specific target RNArecognition, binding and degradation [19]. In recent years RNAi was notonly applied in various biological studies, but applied in therapeuticdevelopment as well [20]. To date, at least 15 therapeutic programsdeveloped from RNAi are in different stages of or have finished clinicaltrials [21].

Delivery Vehicles for siRNA Therapeutics

Even though siRNAs provide a very attractive technology to be developedinto innovative therapeutics, many of the programs didn't succeed.Failed siRNA therapeutic programs either in preclinical studies or inclinical trials have proved that siRNA molecules designed to targetvarious genes could not apply to animals or human beings directlybecause of stability issues [20]. Naked siRNAs have to be modified toprotect them from degradation, or to be packed with other moleculeseither to facilitate cell entry or to be functional to decrease targetgene expression [22]. Therefore, the development of delivery methods hasbeen one of the most important areas in the research and development ofsiRNA therapeutics [23].

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A. HPV Genome and siRNA Designed Targeting Hybrid E7 Gene fromHPV16 and Cotton Rabbit Papilloma Virus (CRPV). The E7 gene is enlargedin the bottom and with the three CRPV motifs marked as red blocks.

FIG. 1B. SiRNA Designed Targeting Wild-type E7 gene of HPV16 and HybridE7 Gene from HPV16 and CRPV. The red blocks are CRPV sequences used toreplace the correspondent HPV16 fragments. The yellow highlights in theCRPV E7 siRNAs reflect the codon optimization results. Figure disclosesSEQ ID NOS 159-165, 22, 166-170, 27-28, and 171, respectively, in orderof appearance.

FIG. 1C. Chimeric human rabbit papilloma virus (cH-RPV) geneconstruction. The 3 epitope sequences A, B and C (corresponding to thered blocks highlighted in FIGS. 1A and 1B) from HPV16 E7 gene wereinserted in the end of CRPV E7 gene in the same reading frame.

FIG. 2. psiCHECK-1 map.

FIG. 3. Rabbit Skin Model of Papilloma Virus Infection.

FIG. 4A. 25 mer siRNA is more potent than 21 mer siRNA.

FIG. 4B. 25 mer siRNA is more potent than 21 mer siRNA.

FIG. 5. HPV16 E7 Expression Knocking Down by siRNAs in SiHa Cells.

FIG. 6. HPV16 E7 Expression Knocking Down by CRPV Hybrid siRNAs in SiHaCells.

FIG. 7. The effect of the first batch siRNAs on knocking-down of HPV18E7mRNA expression.

FIG. 8. The potent siRNAs are capable of reducing the E7 gene expressionin the SiHa cell line. Four siRNAs against the hybrid E7 gene werefurther tested for their ability to reduce the E7 protein expression inSiHa cells. The Western blot (top) and quantitative data (bottom) showedthat the potency of the siRNA reducing the E7 protein expression arelisted in this order, -45>-43>-44>-37, which fit the results in theqRT-PCR experiment.

FIG. 9. Rabbit skin inoculated with different papilloma virus, andtreated with different siRNA-HKP paste (L). Elizabethan collars wereapplied to the animals to prevent disturbance of the treated area by theanimal (R).

FIG. 10. SiRNA (siNA-CRPV-43) treatment inhibited the skin warts growthinduced by cH-RPV in the rabbit model (L), data (R).

FIG. 11. Data summary of the treatment of cH-RPV by different siRNAs.The effective siRNAs are highlighted.

DESCRIPTION OF THE INVENTION

The invention provides siRNA molecules, compositions containing themolecules, and methods for using the molecules and compositions to treatHPV infections and certain other infections, in particular, HIV, HSV,and fungal infections. The compositions include a cocktail of siRNAmolecules targeted against HPV, formulated with dendrimer, a polymerwhich is effective against HIV and HSV infections. The compositions alsoinclude a cocktail of siRNA molecules targeted against HPV, formulatedwith histidine-lysine (HK) polymer, a polymer which has been shown to beeffective in vitro to inhibit fungal infections.

As used herein, an “siRNA molecule” or an “siRNA duplex” is a short,double-stranded oligonucleotide that interferes with the activity of RNAexpressed by a gene in a cell, after the molecule is introduced into thecell. The molecules are constructed by techniques known to those skilledin the art, given the teachings disclosed herein. Such techniques aredescribed in U.S. Pat. Nos. 5,898,031, 6,107,094, 6,506,559, and7,056,704 and in European Pat. Nos. 1214945 and 1230375, which areincorporated herein by reference in their entireties.

The siRNA molecule of the invention is an isolated siRNA molecule thatbinds to a single stranded RNA molecule, which is a messenger RNA (mRNA)that encodes at least part of a peptide or protein of a humanpapillomavirus (HPV). The mRNA can be encoded by any gene in an HPV,including an early or late stage viral proliferation gene. In oneembodiment, the siRNA molecule binds to mRNA molecules from differentspecies of HPV. In one particular embodiment, the HPV species is HPV16,HPV18, HPV6, or HPV11.

In one embodiment, the molecule is an oligonucleotide with a length ofabout 19 to about 35 base pairs. In another embodiment, the molecule isan oligonucleotide with a length of about 19 to about 27 base pairs. Instill another embodiment, the molecule is an oligonucleotide with alength of about 21 to about 25 base pairs. In one particular embodiment,it has 25 base pairs. In all of these embodiments, the molecule may haveblunt ends at both ends, or sticky ends at both ends, or a blunt end atone end and a sticky end at the other. In one particular embodiment, ithas blunt ends at both ends.

The siRNA molecule can be made of naturally occurring ribonucleotides,i.e., those found in living cells, or one or more of its nucleotides canbe chemically modified by techniques known in the art. In addition tobeing modified at the level of one or more of its individualnucleotides, the backbone of the oligonucleotide also can be modified.Additional modifications include the use of small molecules (e.g. sugarmolecules), amino acid molecules, peptides, cholesterol and other largemolecules for conjugation onto the siRNA molecules. Such modificationscan protect the molecule from degradation, improve its potency, reduceits toxicity, and reduce its immune stimulatory effect.

The siRNA molecule may further comprise an immune stimulatory motif.Such motifs can include specific RNA sequences such as 5′-UGUGU-3′(Judge et al., Nature Biotechnology 23, 457-462 (1 Apr. 2005)),5′-GUCCUUCAA-3′ (Hornung et al., Nat. Med. 11, 263-270(2005). See Kim etal., Mol Cell 24; 247-254 (2007). These articles are incorporated hereinby reference in their entireties. These are siRNA sequences thatspecifically activate immune responses through Toll-like receptor (TLR)activation or through activation of key genes such as RIG-I or PKR. Inone embodiment, the motif induces a TH1 pathway immune response. Inanother embodiment, the motif comprises5′-UGUGU-3′,5′-GUCCUUCAA-3′,5′-GGGxGG-3′ (where x is A, T, G and C), orCpG motifs 5′-GTCGTT-3′.

Particular target sequences are shown in Tables 1-4 herein. Thus,certain particular siRNA molecules of the invention bind to and inhibitexpression of one or more of the sequences identified in these tables.Because the siRNA molecules are duplexes, with one strand beingcomplementary to the target mRNA and the other including the samesequence as the target sequence in the mRNA, the sequences shown inTables 1-4 also represent certain specific siRNA molecules of theinvention.

The siRNA molecules of the invention also include ones derived fromthose listed in Tables 1-4. The derived molecules can have less than the25 base pairs shown for each duplex, down to 16 base pairs, so long asthe “core” base pairs remain. That is, once given the specific sequencesshown in the tables, a person skilled in the art can synthesizemolecules that, in effect, “remove” one or more base pairs from eitheror both ends in any order, leaving the remaining contiguous base pairs,creating shorter molecules that are 24, 23, 22, 21, 20, 19, 18, 17, or16 base pairs in length. Thus, the derived molecules consist of: a) 24contiguous base pairs of any one or more of the molecules in Tables 1-4;b) 23 contiguous base pairs of any one or more of the molecules inTables 1-4; c) 22 contiguous base pairs of any one or more of themolecules in Tables 1-4; b) 21 contiguous base pairs of any one or moreof the molecules in Tables 1-4; d) 20 contiguous base pairs of any oneor more of the molecules in Tables 1-4; e) 19 contiguous base pairs ofany one or more of the molecules in Tables 1-4; f) 18 contiguous basepairs of any one or more of the molecules in Tables 1-4; g) 17contiguous base pairs of any one or more of the molecules in Tables 1-4;and h) 16 contiguous base pairs of any one or more of the molecules inTables 1-4. It is not expected that molecules shorter than 16 base pairswould have sufficient activity or sufficiently low off-target effects tobe pharmaceutically useful; however, if any such constructs did, theywould be equivalents within the scope of this invention.

Alternatively, the derived molecules can have more than the 25 basepairs shown for each duplex, so long as the “core” base pairs remain.That is, once given the specific sequences shown in the tables, a personskilled in the art can synthesize molecules that, in effect, “add” oneor more base pairs to either or both ends in any order, creatingmolecules that are 26 or more base pairs in length and containing theoriginal 25 contiguous base pairs.

In one embodiment, the molecule includes a potency enhancer motif. Asused herein, a potency enhancer motif (PEM) is a sequence in the siRNAmolecule that increases the therapeutic effect of the molecule in ananimal model compared to the same molecule without the sequence.Examples of such motifs are ACTCC and GGAGT.

The invention also includes compositions of one or more of the siRNAmolecules. Where there is a plurality of different siRNA molecules, eachone targets a different RNA nucleotide sequence, which can be on thesame RNA target molecule, different RNA target molecules, or anycombination thereof. These compositions, by themselves or in combinationwith pharmaceutically acceptable carriers such as those describedherein, are sometimes called siRNA cocktails. Thus, the inventionprovides multi-targeted siRNA cocktails for the treatment of HPV.

All possible combinations of types of molecules and targets are includedin the invention. For example, the targeted mRNA molecules may encode orregulate the expression of one or more HPV proteins. In one embodiment,the composition comprises two or more different siRNA molecules, eachbinding to a different mRNA target sequence. In another embodiment, thecomposition comprises three different siRNA molecules, each binding to adifferent mRNA target sequence. In still another embodiment, thecomposition comprises more than three different siRNA molecules, eachbinding to a different RNA target sequence. In one embodiment, the siRNAmolecules target one or more of the mRNA sequences that are transcribedfrom one or more of the gene sequences listed in Tables 1-4 herein. Forexample, the cocktail can inhibit expression of one or more of HPV E6,E7, E1, E2, E4, E5, L2, and L1 genes in human tissue. In one embodiment,it inhibits expression of two or more HPV E6, E7, E1, E2, E4, E5, L2,and L1 genes.

As previously mentioned, the siRNA cocktails of the invention comprisetwo or more different siRNA molecules of the invention in apharmaceutically acceptable carrier. Such carriers are generally knownto those skilled in the art and include saline, sugar solutions,polypeptides, polymers, lipids, creams, gels, micelle materials, andmetal nanoparticles.

In one embodiment, the carrier comprises at least one of the following:a glucose solution, a polycationic binding agent, a cationic lipid, acationic micelle, a cationic polypeptide, a hydrophilic polymer graftedpolymer, a non-natural cationic polymer, a cationic polyacetal, ahydrophilic polymer grafted polyacetal, a ligand functionalized cationicpolymer, a ligand functionalized-hydrophilic polymer grafted polymer, aligand functionalized liposome, dendrimer, dendrimer solutions, HKpolymer, and HK polymer solutions. Examples of polymers include abiodegradable histidine-lysine polymer, a biodegradable polyester, suchas poly(lactic acid) (PLA), poly(glycolic acid) (PGA), andpoly(lactic-co-glycolic acid) (PLGA), a polyamidoamine (PAMAM)dendrimer, a cationic lipid, or a PEGylated PEI. Cationic lipids includeDOTAP, DOPE, DC-Chol/DOPE, DOTMA, and DOTMA/DOPE. In still anotherembodiment, the carrier is a histidine-lysine copolymer that forms ananoparticle with the siRNA molecule, wherein the diameter of thenanoparticle is about 100 nm to about 400 nm.

The invention also provides a nanoparticle comprising one or more of thesiRNA molecules of the invention, a pharmaceutically acceptable carrier,such as one or more of those described herein, and a targeting ligand.Examples of such ligands include one or more of an RGD peptide, such asH-ACRGDMFGCA-OH (SEQ ID NO: 2), an RVG peptide, such asH-YTIWMPENPRPGTPCDIFTNSRGKRASNG-OH (SEQ ID NO: 3), or a FROP peptide,such as H-EDYELMDLLAYL-OH (SEQ ID NO: 4).

The molecules and compositions of the invention interfere with viralreplication of HPV. Certain of the compositions also (1) interfere withviral replication of herpes simplex virus (HSV) and humanimmunodeficiency virus (HIV) in mucosal tissues, such as genitaltissues, and (2) treat fungal infections. The molecules and compositionsof the invention are used to treat and/or prevent HPV, HSV, HIV, andfungal infections.

The invention also provides a method of treating a mammal with an HPVinfection by administering to the mammal a therapeutically effectiveamount of one or more of the siRNA molecules of the invention or atherapeutically effective amount of one or more of the compositions ofthe invention. In one embodiment, the mammal is a human, non-humanprimate, or rodent, such as a mouse, rat, or guinea pig. Rodents areparticularly useful for laboratory experiments. In a particularembodiment, the mammal is a human patient.

The compositions are administered by techniques known to those skilledin the art. In one embodiment, the composition comprises at least threesiRNA molecules at a ratio determined by the potency of each siRNAmolecule and the therapeutic needs of the mammal. In another embodiment,the composition comprises three different siRNA molecules at a ratio of1:1:1, 1:1.5:0.5, or 0.5:0.5:2.

Certain compositions of the invention have an anti-HIV and an anti-HSVactivity, and others have an anti-fungal activity. As mentioned above,dendrimer has an anti-HIV and an anti-HSV activity, and HK polymer hasan anti-fungal activity. Therefore, the invention includes a method oftreating a mammal with an HPV infection and with an HIV and/or HSVinfection comprising administering to the mammal a pharmaceuticallyeffective amount of a composition comprising one or more siRNA moleculesof the invention and dendrimer. It further includes a method of treatinga mammal with an HPV infection and a fungal infection comprisingadministering to the mammal a pharmaceutically effective amount of acomposition comprising one or more siRNA molecules of the invention andan HK polymer.

The compositions of the invention also can be used with atherapeutically effective amount of other anti-infective agents.

The siRNA molecules and compositions of the invention can also be usedprophylactically against HPV, HSV, HIV, and fungal infections. Atherapeutically effective amount of one or more of the siRNA moleculesof the invention or a therapeutically effective amount of one or more ofthe compositions of the invention are administered to a mammal. In oneembodiment, the mammal is a human patient.

The following examples illustrate certain aspects of the invention andshould not be construed as limiting the scope thereof.

EXPERIMENTAL DESIGN, TECHNIQUES, AND EXAMPLES Prepare Potent siRNADuplexes Targeted to Genes in HPV16, HPV18, HPV6, and HPV11

In the preliminary studies, we have demonstrated that 25-mer siRNAs aremost potent at inhibiting the expression of a specific gene. To ensurethe potency of each siRNA molecule for the target gene knockdown,several key features should be considered during the in silico designand the later in vitro and in vivo tests so that the siRNAs:

(1) have the optimum thermodynamics for target sequence binding;

(2) have sufficient length for RISC binding;

(3) have eliminated (or added) immune stimulating motifs;

(4) have minimized “Off-Target” potential;

(5) pass through patent searching with no conflict with the currentpatents; and

(6) have no interaction when multiple sequences are mixed in a cocktail.

In this invention, we designed siRNA duplexes targeting the conservedgene sequences shared by as many HPV species as possible to increase theuniversality of the siRNAs. In addition, our preliminary results havedemonstrated that 25mer siRNA is more potent than 21mer siRNA. We use25mer siRNA as our design for the multi-targeted siRNA cocktailtargeting to both early (E6, E7, E1, E2, E4, E5) and late (L2 and L1)genes simultaneously (FIG. 1A). All the gene silencing potencies weretested and validated first in the cell culture experiments by RT-PCR andELISA for efficacy of multi-targeting siRNA on gene knockdown at bothtranscription and translation levels. Once the most effective siRNAduplexes are selected for both early and late genes from the geneknockdown experiments in cell culture models, we further investigatedthe optimal combination of siRNA set as the active pharmaceuticalingredient (API) and tested optimized siRNA combinations in a rabbitmodel.

Applying our siRNA duplexes in cell based high throughput screening, wewere able to select the most effective siRNA molecules to reduce HPV16,HPV18, HPV6, and HPV11 gene expression. The siRNAs target all 8 genes ofE6, E7, E1, E2, E4, E5, L2 and L1 from HPV16, 18, 6 and 11 with some ofthe siRNA incorporating the Potency Enhancer Motif (PEM) sequence. (Seebelow.)

Molecular Design of siRNA Sequence

Table 1 shows the siRNAs designed against the 7 genes from HPV16 (withno qualified siRNAs for the E5 gene). Table 2 shows the siRNAs designedagainst the 8 genes in HPV18. Tables 3 and 4 are the common siRNAs forboth HPV6 and HPV11. Generally, we choose 8 siRNAs for each gene. Ifthere are not enough qualified siRNAs, the number will be less than 8.The genes are arranged according to the transcription starting sites.Each gene also marked the transcription start and stop sites. ThePotency Enhancer Motif (PEM) sequence (GGAGT) and the reversed strand(ACTCC) are highlighted with yellow or green, respectively.

We have designed 8 siRNAs for each gene in HPV strain (selected fromabout 400 siRNA pools for each HPV strain). The following are thespecific sequences of siRNA targeting each gene for each individual HPVstrain. In HPV16, we did not find any qualified siRNA for gene E5 basedon the parameters we set for selection in silico in above section. Inaddition, we found only 7 qualified siRNAs for gene E4. Furthermore,#163 and #167 target both genes of E2 and E4. The overlap has reducedthe total number of unique sequences of siRNA for HPV16 to 53 (Table 1).

In HPV18, there are only 7 and 6 siRNA molecules which are qualified forgene E4 and E5, respectively, based on our selection criteria summarizedin previous section (Table 2). In addition, #173 and #178 target bothgenes E2 and E4. Thus, our total number of unique siRNAs for HPV18 isreduced to 59 (Table 2).

In contrast with HPV16 and 18, where we could not find any siRNAs of25-mer which share the same the common sequence between 16 and 18, inHPV6 and 11, among the siRNAs we have designed (about 250 for each), wehave found 10 common siRNAs for both HPV-6 and HPV-11 as shown in Table3. The 10 common siRNAs target the following genes in both HPV6 and 11:E7, E1, E2, L2 and L1. The common siRNAs found in HPV6 and 11 has madeour selection process simpler and easier. We only need to synthesize the10 siRNAs and test their ability to reduce the expression of HPV6 and 11genes.

In order to screen potent siRNAs in an in vivo rabbit model [24], wehave inserted a codon optimized cotton rabbit papilloma viral (CRPV)gene motif in HPV16 E7, and designed siRNAs to target the CRPV motif andadjacent HPV16 E7 gene (FIGS. 1A and 1B, Table 4). The siRNAs werescreened in SiHa cells to choose the ones which can knock down the E7gene expression.

Potency Enhancer Motif (PEM) Sequence

Table 1 lists the siRNAs designed against the 8 genes from HPV16. Table2 lists the siRNAs designed against the 8 genes in HPV18. Table 3 and 4are the common siRNA molecules targeting both HPV6 and HPV11. The genesare arranged according to the transcription starting sites. Each genealso marked the transcription start and stop sites. The Potency EnhancerMotif (PEM) sequence (GGAGT) and reversed strand (ACTCC) are shown.

We chose 8 siRNAs molecules for each of the 8 genes in both HPV16 andHPV18, with some exceptions (see below). In HPV16, we did not find aqualified siRNA for gene E5 based on the parameters we set for selectionin silico. We found 7 qualified siRNAs for gene E4. Among the 55 siRNAmolecules for HPV16, three of them (#231, #232 and #315) are withPotency Enhancer Motif (PEM) (GGAGU/ACUCC) sequences, and targetinggenes L2 and L1, respectively (Table 1). #163 and #167 target both genesof E2 and E4. The overlap has reduced the total number of uniquesequence of siRNA to 53.

In HPV18, there are 7 and 6 siRNAs qualified for genes E4 and E5,respectively, based on our selection criteria summarized in previoussection (Table 2). In addition, #173 and #178 target both genes E2 andE4. Thus our total number of unique siRNAs for HPV18 is 59 (Table 2).Among the 59 siRNA molecules for HPV 18, 9 of them (#25, $26, #38, #78,#79, #87, #345, #350, and #351) are with Potency Enhancer Motifsequences. Two of the siRNA molecules (#25 and #26) are for E6 gene, one(#38) is for E7 gene, three (#78, #79, and #87) are for E1 gene, andthree (#345, #350 and #351) are for L1 gene (Table 2).

Cloning of 8 Genes (E6, E7, E1, E2, E4, E5, L2, and L1) from HPV16, 18,6 and 11 in Luciferase Expression Vector to Make Fusion Genes

HPV16, 18, 6 and 11 genes were cloned into psiCHECK1/2 vector (FIG. 2.produced by Promega, a dual luciferase system with build-in internalluciferase control system). XhoI and EcoRI restriction enzyme sites wereadded in the 5′ and 3′ and of PCR amplified genes, and fused to theluciferase gene in the same open reading frame (ORF). Each constructharbors one or more genes depending on the size of the genes. Aftercloning, the constructed plasmids were used to transfect HeLa cells andpermanently integrate the genes into the chromosomes to get stable celllines. As an alternative, each construct could also be used in transienttransfection together with the siRNAs for testing of gene expressionknocking down (silencing) by siRNA. After the establishment of the cellline, a time course of Renilla luciferase activity was titrated. Thenthe specific gene knock-down was evaluated by the luciferase expressionlevel. If transient transfection system was used, then co-transfectionor stepwise transfection was applied with siRNA designed for screeningby luciferase assay with proper controls.

Screen siRNAs in Cell Lines Harboring HPV Genes

SiHa is a cervical carcinoma cell line harboring HPV16 genome andexpressing oncoproteins E6 and E7 [25, 26]. SiHa cell line was used toscreen the function of siRNAs targeting E6 and E7 genes in the strain ofHPV16. SiHa cells was grown in RPMI 1640 medium supplemented with 10%FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 100μM non-essential amino acids, 1 mM sodium pyruvate, 50 μM 2-ME, 400μg/ml G418 and 10% NCTC-109 medium at 37° C. with 10% CO₂. The cellswere transfected with siRNAs complexed with LipofectAmine 2000 accordingto manufacturer's instruction. The cells were harvested and qRT-PCR wasapplied to evaluate the gene expression level of E6 and E7. The samecell samples were applied for ELISA and Western analysis.

Similarly, HeLa cervical carcinoma cell which has HPV18 genomeintegrated in the cellular genome [27] was used to screen siRNAstargeting HPV18 genes' expression. The cells were grown in the mediumsimilarly described in above section. The siRNA transfection, qRT-PCR,ELISA and Western were followed the same procedures.

For siRNAs targeting HPV6 and 11, the screen was applied HPV6bcDNA-based C33A (non-cervical carcinoma) cell line [28]. The cells weregrown in the medium described above. The siRNA transfection, qRT-PCR,ELISA, and Western will follow the procedure described in the previoussection.

Validation of siRNA Cocktail in Cell Line

In Vitro Transfection of siRNA

SiHa cells, maintained in Dulbecco's Minimal Essential Medium (DMEM)containing 10% fetal calf serum (FCS) and 20 mM glutamine, were examinedfor the ability of individual siRNAs to knock down particular genetargets. Cells were transfected using a Lipofectamine 2000 (Invitrogen,CA). Briefly, the cells were seeded (1×105 per well) in a 6-well platein 2 ml of DMEM medium. The siRNA was diluted in 0.2 ml serum-free Optimedium mixed with 3 μl of the transfection reagent, incubated for 30 minat room temperature, then added drop-wise into the cell culture. TargetmRNA and protein levels and effects on cells were analyzed in 48 h.

Screening siRNAs Against HPV16, 18, 6 and 11 Genes in HeLa229 byMeasuring Luciferase Strength

HeLa229 cell line (ATCC) has been co-transfected with the plasmidsencoding the fusion gene of HPV viral gene and luciferase and each ofcorrespondent siRNA. In the controls, the vector with only theluciferase, or the fusion gene co-transfected with un-related siRNAswere used. The luciferase strength is measured in all the samples toscreen the most active siRNA molecules which can knock down theexpression of luciferase. After luciferase assays, the same samples wereapplied for RT-PCR, ELISA and Western analysis. After screeninganalysis, two siRNAs for each gene in HPV16, 18, 6 and 11 will beselected for the combination and in vivo studies (below).

Screen siRNAs in Cell Lines Harboring HPV Genes

SiHa is a cervical carcinoma cell line harboring HPV16 genome andexpressing oncoproteins E6 and E7 [25, 26]. SiHa cells were used toscreen the function of siRNAs targeting E6 and E7 genes in the strain ofHPV16. SiHa cells were grown in RPMI 1640 medium supplemented with 10%FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 100μM non-essential amino acids, 1 mM sodium pyruvate, 50 μM 2-ME, 400μg/ml G418 and 10% NCTC-109 medium at 37° C. with 10% CO₂. The cellswere transfected with siRNAs complexed with LipofectAmine 2000 accordingto manufacturer's instruction. The cells were harvested and qRT-PCR wasapplied to evaluate the gene expression level of E6 and E7. The samecell samples were also applied for ELISA and Western analysis.

Similarly, HeLa cervical carcinoma cell, which has HPV18 genomeintegrated in the cellular genome [27], was used to screen siRNAstargeting HPV18 genes' expression. The cells were grown in the mediumsimilarly described in above section. The siRNA transfection, qRT-PCR,ELISA and Western were followed the same procedures.

Gene Knock-Down Assay by RT-PCR Analysis

The gene knockdown results can be evaluated by measuring the mRNAchanges within siRNA treated cells using RT-PCR to amplify RNA isolatedfrom corresponding cells. Selection of the appropriate upstream anddownstream primers is the initial step for evaluation of targeted geneknockdown and choice of the appropriate cell lines. The sequences of theprimers for RT-PCR analysis are:

HPV16 PCR primer list HPV16-1: 16E6-1F (191-461): GGAATCCATATGCTGTATGTPCR length: 270 bp (SEQ ID NO: 5) 16E6-1B (191-461):CTACGTGTTCTTGATGATCT (SEQ ID NO: 6) HPV16-2: 16E6-2F (278-448):CAACATTAGAACAGCAATAC PCR length: 170 bp (SEQ ID NO: 7)16E6-2B (278-448): ATGATCTGCAACAAGACATA (SEQ ID NO: 8) HPV16-E7-1:16E7-1F (21-43): ATTGCATGAATATATGTTAGATT PCR length: 250 bp(SEQ ID NO: 9) 16E7-1B (248-270): CACAATTCCTAGTGTGCCCATTA(SEQ ID NO: 10) HPV18 PCR primer list HPV18-1: 18E6-1F (65-84):ACACTTCACTGCAAGACATA PCR length: 196 (SEQ ID NO: 11) 18E6-1B: (241-260):CCATACACAGAGTCTGAATA (SEQ ID NO: 12) HPV18-2: 18E6-2F (107-126):AGACAGTATTGGAACTTACA PCR length: 151 (SEQ ID NO: 13) 18E6-2B (238-257):TACACAGAGTCTGAATAATG (SEQ ID NO: 14) HPV18-E7-1: 18E7-1F (38-54):TGCATTTAGAGCCCCAA PCR length: 253 (SEQ ID NO: 15) 18E7-1B (275-291):CACAAAGGACAGGGTGT (SEQ ID NO: 16)

Total RNA was extracted from cell culture or tumor tissues with RNeasymini kit (Qiagen, Valencia, Calif.) according to the manufacturer'sinstructions. For RT-PCR, the first cDNA strands were synthesized byusing cDNA Synthesis Kit (GE Healthcare, Chicago, Ill.) according to themanufacturer's instructions. The PCR reaction was started with lowercycle numbers, from 25, 30 to 35 to avoid the possible amplificationplateau. Both Geneamp 9700 Thermalcycler and Taqman (ABI, CA) were usedfor PCR analysis. The amplicons were subjected to the gelelectrophoresis analysis.

ELISA Assay for Luciferase Expression

The HPV gene expression after siRNA knock down could be evaluated by theprotein level assay of Luciferase through Enzyme Linked ImmunosorbentAssay (ELISA), since all the HPV gene were fused with luciferase gene.The same samples were applied for ELISA assays for Luciferase.

ELISA with specific antisera will be used for validation ofsiRNA-mediated down-regulation targeted proteins according to themanufacturer's instructions. Antibodies against Luciferase will bepurchased from Promega.

Western Analysis for Luciferase Expression

The HPV gene expression after siRNA knocking down was evaluated byWestern analysis against HPV16-E7. The same samples used in the aboveassays were applied to Western analysis to evaluate gene down regulationby each specific siRNA molecule. Antibodies against HPV16-E7 werepurchased from Promega.

SiRNA Cocktail

The most potent siRNA to down regulate each gene in HPV will be groupedto evaluate the function to knock-down several genes' expression withineach of species of HPV16, 18, 6 and 11. Following that, a complex toinclude siRNAs targeting all 4 HPV species will be formed to get thefinal cocktail to target either early or late genes in HPV16, 18, 6 and11.

SiRNA Duplex with Potency Enhancer Motif

The siRNA molecules with Potency Enhancer Motif (PEM) will be comparedwith other siRNA molecules in Luciferase assay, RT-PCR, ELISA andWestern.

Selection of siRNAs with and without PEM Sequence

In our siRNA design, L2 and L1 from HPV16 have 2 and 1 siRNAs with PEMsequence, respectively. While in HPV18, E6, E7, E1, and L1 each have 2,1, 3, and 3 siRNAs with PEM sequence (Table 1 and 2). Special attentionwill be paid to those genes and siRNAs. If the in vitro cell screeningshowed acceptable effectiveness of the siRNAs to inhibit designated geneinhibition, those siRNAs will be paired with the ones without PEM buttarget the same gene for further animal studies.

Validation of siRNA Cocktail Complexed with Dendrimer in a Rabbit Model

Dendrimer Selection

The anionic poly-(L-Lysine) dendrimer (PAMAM, the active ingredient ofVivaGel®) was used to complex with selected siRNA duplexes. dendrimeralone or siRNA alone was compared with the complex of dendrimer andsiRNAs at different ratios. Each designed group was applied in thecottontail rabbit model [24].

Complex of siRNAs with Dendrimer

Selected siRNA were formulated with dendrimer in the ratio found above.The testing condition in the animal included the dendrimer alone andsiRNA alone as control. Non-relevant siRNA was also used as control.

Since the active ingredient of VivaGel® is an anionic poly-(L-Lysine),the ratio of siRNA with dendrimer was tested to reach the optimumsurface charge of the complex so that the VivaGel® is still active asproved in other experiments. Non-relevant siRNA with similar charge wasformulated with VivaGel® as control.

Therapeutic Program Verses Prophylactic Program

In the therapeutic program, the siRNA was applied on the animal skinafter the virus was inoculated. While in the prophylactic program, siRNAwas applied prior to the inoculation of the HPV virus. The time intervalwas tested to find the best time point to apply “prophylactic” siRNAs onthe animals before the virus challenge.

Specifically, for example, if siRNA #231 and 232 (each with the PEMsequence) and #194 and 258 (both without PEM sequence) in HPV16 showedeffectiveness in inhibiting HPV16 L2 gene expression in cell screening,the pair of #231 and 194, or the pair of #232 and 258, or the pair of#231 and 258, or the pair of #232 and 194 will be selected for furthertherapeutic and prophylactic studies. In both studies, each pair wasapplied on one rabbit. The siRNA with PEM (e.m. #231) and without PEM(e.m. #194) was applied on left and right side of the animalrespectively, with increased dose (from 5 μg to 100 μg), and propercontrol. The virion particle was inoculated on the same spot prior tothe siRNA for the treatment program, or after the application of siRNAfor prophylactic program. The interval between two treatments was testedin a separate experiment to cover from 4 hours to 72 hours.

Chimeric HPV Virion Particle Preparation

Using pseudovirion technology, we produced infectious particles composedof a heterologous cottontail rabbit Papilloma virus (CRPV) genomeencapsulated with HPV16/18 or CRPV capsid proteins [29]. The CRPV genomecontains the simian virus 40 (SV40) origin of replication (337 bp)cloned at the BglII site within the upstream regulatory region forimproved CRPV genome amplification in the packaging cell line 293TT.Both homologous (CRPV capsid/CRPV genome) and chimeric (HPV16 or 18capsid/CRPV genome) Papilloma virion particles will be preparedaccording to the methods [29]. The virion particle prepared this way isinfectious in vitro at low volume (<10 μl) to induce epidermal papillomaon New Zealand White rabbits [24].

Rabbit Skin Inoculation of HPV Virus and Treat with Complex of siRNA andDendrimer

We used the established cottontail rabbit model [24] to test theefficacy of our siRNA and the complex of siRNA and dendrimer to inhibitthe lesion formation on the animal skin (FIG. 3). Briefly, the siRNAs,dendrimer, and the formulated complex of siRNA and dendrimer wereapplied on three groups of six rabbit each in various amounts (5 μg to100 μg), before or after the virus inoculation depending on the programused to test the efficacy. Four days after the final treatment, bloodsamples were collected for analysis of capsid-specific antibody by usingan enzyme-linked immunosorbent assay (ELISA). Each serum sample wastested for the ability to bind CRPV, HPV16 and HPV18 L1-only virus-likeparticles (VLPs) adhered overnight on 96-well plates in PBS (pH 7.4) at4° C.

Validation of siRNA Cocktail Complexed with HK Polymer in a Rabbit Model

HK Polymer Selection

H2K4b is a histidine-lysine polymer with MW of 11,137, and showed thehighest activity to inhibit the growth of fugal strains, C. albicans andC. kefyr [18]. The structure of H2K4b is shown here:

KKK(KHKHHKHHKHHKHHKHHKHK)4 (core sequence disclosed as SEQ ID NO: 1)Because of its anti-fungus activity, H2K4b will be selected as the HKpolymer to formulate with the designed siRNAs against HPV16, 18, 6 and11.Complex of siRNAs with HK Polymer

Selected siRNA were formulated with HK polymer in the ratio found asdescribed above. The testing condition in the animal included the HKpolymer alone and siRNA alone as a control. Non-relevant siRNA was alsoused as a control.

The ratio of siRNA with HK Polymer was tested to reach the optimumsurface charge for efficient cell delivery. Non-relevant siRNA withsimilar charge was formulated with HK polymer as control.

Rabbit Skin Inoculation of HPV Virus and Treat with Complex of siRNA andHK Polymer

We used the established cottontail rabbit model [24] to test theefficacy of our siRNA and the complex of siRNA and HK polymer to inhibitthe lesion formation on the animal skin (FIG. 3). Briefly, the siRNAs,HK polymer, and the formulated complex of siRNA and HK polymer wereapplied on three groups of six rabbit each in various amounts (5 μg to100 μg), before or after the virus inoculation, depending on whatprogram were used to test the efficacy. Four days after the finaltreatment, blood samples were collected for analysis of capsid-specificantibody by using an enzyme-linked immunosorbent assay (ELISA). Eachserum sample was tested for the ability to bind CRPV, HPV16 and HPV18L1-only virus-like particles (VLPs) adhered overnight on 96-well platesin PBS (pH 7.4) at 4° C.

Example 1 25 mer siRNA is More Potent than 21 mer siRNA

Although initial studies mostly applied 19mer and 21mer siRNA duplexes,several lines of evidence showed that 23mer, 25mer and 27mer siRNAduplexes exhibited more potent silencing effects than 19mer and 21mersiRNAs did. We found that 25mer duplexes with blunt ends are the mostpotent inhibitors. We have tested a 25mer siRNA duplex targeting humanVEGF gene, hVEGF-25c (sense: 5′-CACAACAAAUGUGAAUGCAGACCAA-3′ (SEQ ID NO:17); Antisense: 5′-UUGGUCUGCAUUCACAUUUGUUGUG-3′ (SEQ ID NO: 18)),comparing it to a 21mer siRNA duplex, which has been tested many timesas one of the most potent VEGF specific inhibitory duplexes, hVEGF-21a(sense: 5′-UCGAGACCCUGGUGGACAUTT-3′ (SEQ ID NO: 19); antisense:5′-AUGUCCACCAGGGUCUCGATT-3′ (SEQ ID NO: 20)), in the cell culturefollowed with Q-RT-PCR analysis (FIG. 4A). A similar study also wascarried out with ELISA analysis for the difference between the 25 merand 21 mer siRNA both targeting VEGF (FIG. 4B).

Example 2 Cell Lines and PCR Primers for Potent siRNA Screening

SiHa cells which host HPV16 genes was chosen as the in vitro cell modelto screen siRNAs against E7 gene expression in both HPV16 and chimericalhuman rabbit papilloma virus. Transfected SiHa cells were also appliedin Western to evaluate the E7 protein expression. HeLa cells which hostHPV18 genes was chosen as the in vitro cell model to screen siRNAsagainst E7 gene expression in HPV18. Quantitative RT-PCR was used toevaluate the E7 gene expression for potent siRNA screening.

PCR primers used to monitor the E7 gene expression in SiHa cell line:

16E7-Forward: ATTGCATGAATATATGTTAGATT (SEQ ID NO: 9) 16E7-Reverse:CACAATTCCTAGTGTGCCCATTA; (SEQ ID NO: 10)

PCR primers used to monitor the E7 gene expression in HeLa cell line:

18E7-1Forward: TGCATTTAGAGCCCCAA (SEQ ID NO: 15) 18E7-1Reverse:CACAAAGGACAGGGTGT. (SEQ ID NO: 16)

Example 3 Design and Screen Small Interfering RNAs (siRNAs) Against E7Gene in HPV16

The following are the DNA sequences in HPV16 E7 gene corresponding toeach of the siRNA molecule. They all qualified in our in silica designsystem, and used as the model for siRNA syntheses.

Rec#, 34, GCATGGAGATACACCTACATTGCAT, (SEQ ID NO: 21) Rec#, 37,GGACAGAGCCCATTACAATATTGTA, (SEQ ID NO: 22) Rec#, 39,GCAAGTGTGACTCTACGCTTCGGTT, (SEQ ID NO: 23) Rec#, 40,GCGTACAAAGCACACACGTAGACAT, (SEQ ID NO: 24) Rec#, 41,CGTACAAAGCACACACGTAGACATT, (SEQ ID NO: 25) Rec#, 42,GCACACACGTAGACATTCGTACTTT, (SEQ ID NO: 26) Rec#, 43,GGAAGACCTGTTAATGGGCACACTA, (SEQ ID NO: 27) Rec#, 44,CCTGTTAATGGGCACACTAGGAATT, (SEQ ID NO: 28)

Through the in vitro SiHa cell screening, it showed that 4 out of 8siRNAs reduced the E7 gene expression over 80% (FIG. 4).

Example 4 Construction of Chimerical Human Rabbit Papilloma Virus(cH-RPV)

Three epitopes (A, B and C in FIG. 5 top) from HPV16 E7 gene wereapplied to construct hybrid E7 gene between CRPV and HPV16. Epitope Acorresponds to amino acid 82 to 90, while Epitope B corresponds to aminoacid 49 to 57, and Epitope C corresponds to amino acid 11 to 20. Thesequences were inserted at the end of CRPVE7 before the stop codon inthe same open reading frame (ORF) to form a hybrid protein of E7 asshown (FIG. 5, bottom).

The potency of each of the hybrid viruses to form papilloma wasconfirmed in the rabbit skin.

Example 5 Design and Screen Small Interfering RNAs (siRNAs) Against E7Gene in cH-RPV (Chimerical Human Rabbit Papilloma Virus)

CRPE7-36 5′- GCAUGAAUAUAUGUUGGAUCUGCA-3′ (SEQ ID NO: 29) CRPE7-375′- GGACAGAGCCCACUACAACAUCGU-3′ (SEQ ID NO: 30) CRPE7-385′- GCCCACUACAACAUCGUGACCUUUU-3′ (SEQ ID NO: 31) CRPE7-435′- GGAAGACCUGCUGAUGGGCACCCU-3′ (SEQ ID NO: 32) CRPE7-445′- CCUGCUGAUGGGCACCCUGGGCAU-3′ (SEQ ID NO: 33) CRPE7-455′- GCACCCUGGGCAUCCUGUGCCCCAU-3′ (SEQ ID NO: 34)

Six siRNA shown above designed to target the three epitopes in thehybrid E7 gene, with CRPE7-36 targeting Epitope C, CRPE7-37 and CRPE7-38targeting Epitope B, and CRPE7-43, -44 and -45 targeting Epitope A.

The results in FIG. 6 have shown that 4 of the hybrid siRNAs were ableto reduce E7 gene expression over 60%, with #45 as the most potent siRNAwhich knocked-down E7 gene expression over 80%. The potency is listedas, 45 (0.197)>43 (0.305)>44 (0.350)>37 (0.383). The numbers inparentheses are the expression of E7 gene comparing with negativecontrol.

Example 6 Design Small Interfering RNAs (siRNAs) Against E7 Gene inHPV18

The following are the siRNA sequences against corresponding sequences inHPV18 E7 gene. They all qualified in our in silica design system.

HPV18E7-31: 5′- GCAUGGACCUAAGGCAACAUUGCAA-3′ (SEQ ID NO: 35) HPV18E7-34:5′- GGUUGACCUUCUAUGUCACGAGCAA-3′ (SEQ ID NO: 36) HPV18E7-36:5′- GCAAUUAAGCGACUCAGAGGAAGAA-3′ (SEQ ID NO: 37) HPV18E7-38:5′- CGAUGAAAUAGAUGGAGUUAAUCAU-3′ (SEQ ID NO: 38) HPV18E7-39:5′- CGAGCCGAACCACAACGUCACACAA-3′ (SEQ ID NO: 39) HPV18E7-43:5′- GCCAGAAUUGAGCUAGUAGUAGAAA-3′ (SEQ ID NO: 40) HPV18E7-44:5′- GCUCAGCAGACGACCUUCGAGCAUU-3′ (SEQ ID NO: 41) HPV18E7-46:5′- GCUGUUUCUGAACACCCUGUCCUUU-3′ (SEQ ID NO: 42)

Through the in vitro HeLa cell screening, it showed that 3 out of 8siRNAs (#31, #36, and #38) are able to reduce the E7 gene expressionover 50% (FIG. 7).

Example 7 Screen Small Interfering RNAs (siRNAs) Against E7 Gene inHPV18

FIG. 7 shows the effect of the designed siRNAs on knocking-down ofHPV18E7 mRNA expression. The transfections was done on HeLa cells, using40 nM as the action concentration, and the 80 nM negative control siRNAas the NC group. β-actin was used as the internal control gene. Mean±SD.

The results demonstrated that three siRNAs (31#, 36#, 38#) showed fineknocking-down effect (>50%) targeting HPV16E7 mRNA expression. Thesethree siRNAs were applied for the EC50 experiment.

Example 8 Western Analysis to Confirm the Knocking Down of E7 ProteinExpression by Potent siRNAs

Four siRNAs against the hybrid E7 gene were further tested for theirability to reduce the E7 protein expression in SiHa cells by Westernanalysis. In FIG. 8, The Western blot (top) and quantitative data(bottom) showed that the potency of the siRNA reducing the E7 proteinexpression are listed in this order, -45>-43>-44>-37, which fit theresults in the qRT-PCR.

Example 9 Testing the Efficacy of the siRNAs in Skin Infection RabbitAnimal Model (SIRAM)

CRPV/NZW rabbit in the testing In order to test the therapeutic efficacyof the siRNA we have confirmed with our in vitro cell screening system,6 different wild type and hybrid viruses were inoculated on the NZWrabbit skin as illustrated in FIG. 9. Elizabeth collars (FIG. 9, right)were worn by each animal to avoid disturbing of the treated area by theanimals.

In the pilot study six animals were used in the experiments. In eachanimal, L1-R1, L2-R2, L3-R3, L4-R4, L5-R5 and L6-R6 were challenged withsix different constructs respectively as illustrated in the FIG. 7legends. Two weeks after the challenge, the left sites of the papillomaswere treated with corresponding testing siRNAs, N.C siRNA and Cidovofirtopically for five consecutive days. Papilloma outgrowth began to bemonitored at week 3 and until the termination of the experiment at theend of week 5. Pictures were also taken for record. Right sites areuntreated control for the left treated sites. If the siRNA is effective,we should see smaller or no papillomas on the left sites but not on theright sites. The construct that infected L5-R5 sites is more vigorousthan those on sites L2-R2, L3-R3 and L4-R4. L1-R1 infected withwild-type CRPV is used as a specificity control for the SiRNA.Therefore, if an epitope specific siRNA is effective, it should notinfluence L1-R1 sites but the sites that challenged with the constructscontaining this epitope such as L5-R5.

The viruses are described in the following:

L1-R1, wt CRPV DNA 5 ug/site;

L2-R2, CRPV with HPV16E7/A 82-90 insert;

L3-R3, CRPV with HPV16E7/B 45-57 insert;

L4-R4, CRPV with HPV16E7/C 11-20 insert;

L5-R5, CRPV with HPV16 E7/82-90 at L2 insert;

L6-R6, CRPV tandem repeat with HPV16E7.

After papilloma appeared on the skin, we applied different siRNAs to thepapilloma to evaluate the efficacy of them 2 weeks after the viralchallenge. The following are the siRNAs applied on the animals:

Rabbit #3270, siRNA-CRPC-37;

Rabbit #3271, siRNA-CRPC-43;

Rabbit #3272, siRNA-CRPC-44;

Rabbit #3273, siRNA-CRPC-45;

Rabbit #3274, siRNA-NC;

Rabbit #3275, Cidofovir, the positive control;

In one of the examples, CRPV-43 treatment inhibited the papilloma growth(FIG. 10).

The ability of the siRNAs against the hybrid human rabbit papillomagrowth was summarized in FIG. 11.

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All publications, including issued patents and published patentapplications, and all database entries identified by url addresses oraccession numbers are incorporated herein by reference in theirentirety.

Although this invention has been described in relation to certainembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details described herein may be varied considerably withoutdeparting from the basic principles of the invention.

TABLE 1 HPV16 Sequences Corresponding to  the siRNAs Against 8 Genes(Potency Enhancer Motif, PEM,  is underlined and in bold font)HPV16 - 8siRNA Gene = “E6” (83 → 559)   #8, CCCACAGGAGCGACCCAGAAAGTTA(SEQ ID NO: 43)  #10, CGACCCAGAAAGTTACCACAGTTAT (SEQ ID NO: 44)  #11,CCACAGTTATGCACAGAGCTGCAAA (SEQ ID NO: 45)  #15,CGACGTGAGGTATATGACTTTGCTT (SEQ ID NO: 46)  #19,GGGAATCCATATGCTGTATGTGATA (SEQ ID NO: 47)  #22,GCAATACAACAAACCGTTGTGTGAT (SEQ ID NO: 48)  #28,CGGTGGACCGGTCGATGTATGTCTT (SEQ ID NO: 49)  #30,CGATGTATGTCTTGTTGCAGATCAT (SEQ ID NO: 50) Gene = “E7” (562 → 858)  #34,GCATGGAGATACACCTACATTGCAT (SEQ ID NO: 21)  #37,GGACAGAGCCCATTACAATATTGTA (SEQ ID NO: 22)  #39,GCAAGTGTGACTCTACGCTTCGGTT (SEQ ID NO: 23)  #40,GCGTACAAAGCACACACGTAGACAT (SEQ ID NO: 24)  #41,CGTACAAAGCACACACGTAGACATT (SEQ ID NO: 25)  #42,GCACACACGTAGACATTCGTACTTT (SEQ ID NO: 26)  #43,GGAAGACCTGTTAATGGGCACACTA (SEQ ID NO: 27)  #44,CCTGTTAATGGGCACACTAGGAATT (SEQ ID NO: 28) Gene = “E1” (865 → 2813)  #51,GGGTACGGGATGTAATGGATGGTTT (SEQ ID NO: 51)  #64,CGGGTATGGCAATACTGAAGTGGAA (SEQ ID NO: 52)  #81,GCTGCATTTGGACTTACACCCAGTA (SEQ ID NO: 53)  #92,GGAGACACGCCAGAATGGATACAAA (SEQ ID NO: 54)  #97,GGATTGTGCAACAATGTGTAGACAT (SEQ ID NO: 55) #107,GGTGCAGCTAACACAGGTAAATCAT (SEQ ID NO: 56) #118,GGATGTAAAGCATAGACCATTGGTA (SEQ ID NO: 57) #130,CGATGGAGACTCTTTGCCAACGTTT (SEQ ID NO: 58) Gene = “E2” (2755 → 3852)#131, GGAGACTCTTTGCCAACGTTTAAAT (SEQ ID NO: 59) #135,GCTATTTATTACAAGGCCAGAGAAA (SEQ ID NO: 60) #146,GGAAGTGCAGTTTGATGGAGACATA (SEQ ID NO: 61) #151,GGGTCAAGTTGACTATTATGGTTTA (SEQ ID NO: 62) #155,GGAAGTTCATGCGGGTGGTCAGGTA (SEQ ID NO: 63) #163,CGACCCATACCAAAGCCGTCGCCTT (SEQ ID NO: 64) #167,CCAAGATCAGAGCCAGACACCGGAA (SEQ ID NO: 65) #172,GGCATTGGACAGGACATAATGTAAA (SEQ ID NO: 66) Gene = “E4” (3332 → 3619)#158, GCAACGAAGTATCCTCTCCTGAAAT (SEQ ID NO: 67) #159,CGAAGTATCCTCTCCTGAAATTATT (SEQ ID NO: 68) #163,CGACCCATACCAAAGCCGTCGCCTT (SEQ ID NO: 64) #164,GCCGTCGCCTTGGGCACCGAAGAAA (SEQ ID NO: 69) #165,GGCACCGAAGAAACACAGACGACTA (SEQ ID NO: 70) #166,GCACCGAAGAAACACAGACGACTAT (SEQ ID NO: 71) #167,CCAAGATCAGAGCCAGACACCGGAA (SEQ ID NO: 65) Gene = “E5” (3863 → 4099)No sequence can be qualified. Gene = “L2” (4235 → 5656) #192,CGTGCATCGGCTACCCAACTTTATA (SEQ ID NO: 72) #202,GGGTACAGGCGGACGCACTGGGTAT (SEQ ID NO: 73) #217,CCCAGATGTATCAGGATTTAGTATT (SEQ ID NO: 74) #225,CGCCCAGTGGCACGCCTAGGATTAT (SEQ ID NO: 75) #232, CC ACTCCCACTAAACTTATTACATA (SEQ ID NO: 76) #247, GCAGCCTCACCTACTTCTATTAATA(SEQ ID NO: 77) #259, CCAGGGTCTCCACAATATACAATTA (SEQ ID NO: 78) Gene =“L1” (5559 → 7154) #265, GGCTGCCTAGTGAGGCCACTGTCTA (SEQ ID NO: 79) #276,GCTGGTTTGGGCCTGTGTAGGTGTT (SEQ ID NO: 80) #283,GCAGCAAATGCAGGTGTGGATAATA (SEQ ID NO: 81) #289,CCCATGTACCAATGTTGCAGTAAAT (SEQ ID NO: 82) #301,GGGTCTACTGCAAATTTAGCCAGTT (SEQ ID NO: 83) #327,GGAGGCACACTAGAAGATACTTATA (SEQ ID NO: 84) #345,CCTCATCTACCTCTACAACTGCTAA (SEQ ID NO: 85)

TABLE 2 HPV18 Sequences Corresponding to the siRNAs Against 8 Genes(Potency Enhancer Motif, PEM, is underlined and in bold font)HPV18 - 8siRNA Gene = “E6” (105-581)   #6, GCAAGACATAGAAATAACCTGTGTA(SEQ ID NO: 86)   #7, CCTGTGTATATTGCAAGACAGTATT (SEQ ID NO: 87)  #12,CCCATGCTGCATGCCATAAATGTAT (SEQ ID NO: 88)  #18,GGTGCCTGCGGTGCCAGAAACCGTT (SEQ ID NO: 89)  #21,CCAGAAACCGTTGAATCCAGCAGAA (SEQ ID NO: 90)  #24,GGGCACTATAGAGGCCAGTGCCATT (SEQ ID NO: 91)  #25, CCGAGCACGACAGGAACG ACTCCAA (SEQ ID NO: 92)  #26, GGAACG ACTCC AACGACGCAGAGAA (SEQ ID NO: 93)Gene = “E7” (590-907)  #31, GCATGGACCTAAGGCAACATTGCAA (SEQ ID NO: 94) #34, GGTTGACCTTCTATGTCACGAGCAA (SEQ ID NO: 95)  #36,GCAATTAAGCGACTCAGAGGAAGAA (SEQ ID NO: 96)  #39,CGAGCCGAACCACAACGTCACACAA (SEQ ID NO: 97)  #43,GCCAGAATTGAGCTAGTAGTAGAAA (SEQ ID NO: 98)  #44,GCTCAGCAGACGACCTTCGAGCATT (SEQ ID NO: 99)  #46,GCTGTTTCTGAACACCCTGTCCTTT (SEQ ID NO: 100) Gene = “E1” (914-2887)  #50,GGGCACGGGTTGTAACGGCTGGTTT (SEQ ID NO: 101)  #69,GGCAATGTATGTAGTGGCGGCAGTA (SEQ ID NO: 102)  #78, GGGTTACAGCTATATTT GGAGTAAA (SEQ ID NO: 103)  #79, GCTATATTT GGAGT AAACCCAACAA (SEQ ID NO: 104)#104, CCTTATTAGCAGACAGCAACAGCAA (SEQ ID NO: 105) #130,CGTGTTGGACATACTTTGATACCTA (SEQ ID NO: 106) #142,GGAAGAGGAAGATGCAGACACCGAA (SEQ ID NO: 107) Gene = “E2” (2817-3914) #144,CGAAGGAAACCCTTTCGGAACGTTT (SEQ ID NO: 108) #151,GCAAGGGAACATGGCATACAGACAT (SEQ ID NO: 109) #158,GGAATACAGAACCTACTCACTGCTT (SEQ ID NO: 110) #163,GGACAGTGTGTATTATATGACTGAT (SEQ ID NO: 111) #173,CGGTATCCGCTACTCAGCTTGTTAA (SEQ ID NO: 112) #178,GCATTGTGGACCTGTCAACCCACTT (SEQ ID NO: 113) #182,GGTAACACTACGCCTATAATACATT (SEQ ID NO: 114) #187,GGAATACTGACTGTAACATACCATA (SEQ ID NO: 115) Gene = “E4” (3418-3684) #172,CGACACGGTATCCGCTACTCAGCTT (SEQ ID NO: 116) #173,CGGTATCCGCTACTCAGCTTGTTAA (SEQ ID NO: 112) #174,GGTATCCGCTACTCAGCTTGTTAAA (SEQ ID NO: 117) #175,CGCTACTCAGCTTGTTAAACAGCTA (SEQ ID NO: 118) #178,GCATTGTGGACCTGTCAACCCACTT (SEQ ID NO: 113) #179,CCACTTCTCGGTGCAGCTACACCTA (SEQ ID NO: 119) #181,CGGAAACTCTGTAGTGGTAACACTA (SEQ ID NO: 120) Gene = “E5” (3936-4157) #193,GCCATCTGTCTGTATGTGTGCGTAT (SEQ ID NO: 121) #194,GCATGGGTATTGGTATTTGTGTATA (SEQ ID NO: 122) #197,CCCTGCCACAGCATTCACAGTATAT (SEQ ID NO: 123) #198,GCCACAGCATTCACAGTATATGTAT (SEQ ID NO: 124) #199,CCACAGCATTCACAGTATATGTATT (SEQ ID NO: 125) #201,GCCCATGTTACTATTGCATATACAT (SEQ ID NO: 126) Gene = “L2” (4244-5632) #206,GCAAACGGGCTTCGGTAACTGACTT (SEQ ID NO: 127) #221,GGGTACATTCCATTGGGTGGGCGTT (SEQ ID NO: 128) #230,GGGTTTGATATAACATCTGCGGGTA (SEQ ID NO: 129) #242,CCCTACATCTGGAACACATGGGTAT (SEQ ID NO: 130) #253,CCTACCAACAAGTGTCAGTGGCTAA (SEQ ID NO: 131) #262,GCAACTATGTTTACCCGCAGCGGTA (SEQ ID NO: 132) #268,CGGAGGACAATGACTTGTTTGATAT (SEQ ID NO: 133) #280,CCTCCTCTTGGGATGTGCCTGTATA (SEQ ID NO: 134) Gene = “L1” (5430-7136) #288,CCTGCCTCTACACAGTATATTGGTA (SEQ ID NO: 135) #307,GGGTGCAGTTACCTGACCCAAATAA (SEQ ID NO: 136) #333,GGATATGGTGCCATGGACTTTAGTA (SEQ ID NO: 137) #345, CCTCTG ACTCCCAGTTGTTTAATAA (SEQ ID NO: 138) #350, GGTAGATACC ACTCC CAGTACCAAT(SEQ ID NO: 139) #351, CC ACTCC CAGTACCAATTTAACAAT (SEQ ID NO: 140)#364, CCAACTACTAGTTTGGTGGATACAT (SEQ ID NO: 141) #379,CCACTACGTCTTCTAAACCTGCCAA (SEQ ID NO: 142)

TABLE 3 HPV6 and HPV11 Common Sequences Corresponding to siRNAs(Potency Enhancer Motif, PEM, is underlined and in bold font)siRNAs common to HPV6 & 11 Gene = “E7” (530-826)  #3,CCTGTTGCTGTGGATGTGACAGCAA (SEQ ID NO: 143) Gene = “E1” (832-2781)  #8,GGACAGTGGATATGGCTATTCTGAA (SEQ ID NO: 144) #11,CGAGGAAGATGGAAGCAATAGCCAA (SEQ ID NO: 145) #12,GGAAGCAATAGCCAAGCGTTTAGAT (SEQ ID NO: 146) Gene = “E2” (2723-3826) #14,GGAAGTATGTTATGGCAGCACAGTT (SEQ ID NO: 147) Gene = “L2” (4417-5784) #15,CCCTTTAGTCCTGTA ACTCC TGCTT (SEQ ID NO: 148) #16, CCTTTAGTCCTGTA ACTCCTGCTTT (SEQ ID NO: 149) #17, CCTGCTTTACCTACAGGCCCTGTTT (SEQ ID NO: 150)Gene = “L1” (5771-7276) #21, GGCGGCCTAGCGACAGCACAGTATA (SEQ ID NO: 151)#22, GCGGCCTAGCGACAGCACAGTATAT (SEQ ID NO: 152)

TABLE 4 siRNAs against HPV16 E7 and hybrid CRPV E7 16E7-36:5′-GCAUGAAUAUAUGUUAGAUUUGCAA-3′ (SEQ ID NO: 153) CRPE7-36:5′-GCAUGAAUAUAUGUUGGAUCUGCA-3′ (SEQ ID NO: 29) 16E7-37:5′-GGACAGAGCCCAUUACAAUAUUGUA-3′ (SEQ ID NO: 154) CRPE7-37:5′-GGACAGAGCCCACUACAACAUCGU-3′ (SEQ ID NO: 30) 16E7-38:5′-GCCCAUUACAAUACCGUAACCUUUU-3′ (SEQ ID NO: 155) CRPE7-38:5′-GCCCACUACAACAUCGUGACCUUUU-3′ (SEQ ID NO: 31) 16E7-43:5′-GGAAGACCUGUUAAUGGGCACACUA-3′ (SEQ ID NO: 156) CRPE7-43:5′-GGAAGACCUGCUGAUGGGCACCCU-3′ (SEQ ID NO: 32) 16E7-44:5′-CCUGUUAAUGGGCACACUAGGAAUU-3′ (SEQ ID NO: 157) CRPE7-44:5′-CCUGCUGAUGGGCACCCUGGGCAU-3′ (SEQ ID NO: 33) 16E7-45:5′-GCACACUAGGAAUUGUGUGCCCCAU-3′ (SEQ ID NO: 158) CRPE7-45:5′-GCACCCUGGGCAUCCUGUGCCCCAU-3′ (SEQ ID NO: 34)

What is claimed is:
 1. A composition comprising at least two blunt-endedsiRNA molecules and a pharmaceutically acceptable carrier, wherein saidsiRNA molecules are selected from the group consisting of:5'- GGACAGAGCCCAUUACAAUAUUGUA-3' (SEQ ID NO: 154)5'- GCGUACAAAGCACACACGUAGACAU-3' (Corresponding to SEQ ID NO: 24)5'- CGUACAAAGCACACACGUAGACAUU-3' (Corresponding to SEQ ID NO: 25)5'- GCACACACGUAGACAUUCGUACUUU-3' (Corresponding to SEQ ID NO: 26)5'- CCUGUUAAUGGGCACACUAGGAAUU-3' (Corresponding to SEQ ID NO: 28)5'- GGACAGAGCCCACUACAACAUCGU-3' (SEQ ID NO: 30)5'- GGAAGACCUGCUGAUGGGCACCCU-3' (SEQ ID NO: 32)5'- CCUGCUGAUGGGCACCCUGGGCAU-3' (SEQ ID NO: 33)5'- GCACCCUGGGCAUCCUGUGCCCCAU-3' (SEQ ID NO: 34)5'- CGAGCCGAACCACAACGUCACACAA-3' (SEQ ID NO: 39)5'- GCUCAGCAGACGACCUUCGAGCAUU-3' (SEQ ID NO: 41) and5'- GCUGUUUCUGAACACCCUGUCCUUU-3' (SEQ ID NO: 42)

and wherein said carrier comprises a dendrimer or a histidine-lysinepolymer.
 2. The composition of claim 1, wherein the pharmaceuticallyacceptable carrier comprises a histidine-lysine polymer.
 3. Thecomposition of claim 2, wherein the siRNA molecules and thehistidine-lysine polymer form a nanoparticle whose diameter is 100-400nm.
 4. The composition of claim 1, wherein the pharmaceuticallyacceptable carrier comprises a dendrimer.
 5. A method for treating ahuman with an HPV infection comprising administering to said human apharmaceutically effective amount of the composition of claim
 1. 6. Amethod for treating a human with an HPV infection and an HIV and/or anHSV infection comprising administering to said human a pharmaceuticallyeffective amount of the composition of claim
 4. 7. A method for treatinga human with an HPV infection and a fungal infection comprisingadministering to said human a pharmaceutically effective amount of thecomposition of claim
 2. 8. A composition comprising at least threeblunt-ended siRNA molecules and a pharmaceutically acceptable carrier,wherein said siRNA molecules are selected from the group consisting of:5'- GGACAGAGCCCAUUACAAUAUUGUA-3' (SEQ ID NO: 154)5'- GCGUACAAAGCACACACGUAGACAU-3' (Corresponding to SEQ ID NO: 24)5'- CGUACAAAGCACACACGUAGACAUU-3' (Corresponding to SEQ ID NO: 25)5'- GCACACACGUAGACAUUCGUACUUU-3' (Corresponding to SEQ ID NO: 26)5'- CCUGUUAAUGGGCACACUAGGAAUU-3' (Corresponding to SEQ ID NO: 28)5'- GGACAGAGCCCACUACAACAUCGU-3' (SEQ ID NO: 30)5'- GGAAGACCUGCUGAUGGGCACCCU-3' (SEQ ID NO: 32)5'- CCUGCUGAUGGGCACCCUGGGCAU-3' (SEQ ID NO: 33)5'- GCACCCUGGGCAUCCUGUGCCCCAU-3' (SEQ ID NO: 34)5'- CGAGCCGAACCACAACGUCACACAA-3' (SEQ ID NO: 39)5'- GCUCAGCAGACGACCUUCGAGCAUU-3' (SEQ ID NO: 41) and5'- GCUGUUUCUGAACACCCUGUCCUUU-3' (SEQ ID NO: 42)

and wherein said carrier comprises a dendrimer or a histidine-lysinepolymer.
 9. The composition of claim 8, wherein the pharmaceuticallyacceptable carrier comprises a dendrimer.
 10. The composition of claim8, wherein the pharmaceutically acceptable carrier comprises ahistidine-lysine polymer.
 11. The composition of claim 8, wherein saidcomposition comprises three siRNA molecules at a ratio of 1:1:1,1:1.5:0.5, or 0.5:0.5:2.
 12. A method of treating a human with an HPVinfection comprising administering to said human a pharmaceuticallyeffective amount of the composition of claim
 8. 13. A method of treatinga human with an HPV infection and with an HIV and/or HSV infectioncomprising administering to said human a pharmaceutically effectiveamount of the composition of claim
 9. 14. A method of treating a humanwith an HPV infection and with a fungal infection comprisingadministering to said human a pharmaceutically effective amount of thecomposition of claim
 10. 15. A nanoparticle comprising the siRNAmolecules of claim 1, a pharmaceutically acceptable carrier, and atargeting ligand.
 16. A nanoparticle comprising the siRNA molecules ofclaim 8, a pharmaceutically acceptable carrier, and a targeting ligand.17. The composition of claim 10, wherein the siRNA molecules and thehistidine-lysine polymer form a nanoparticle whose diameter is 100-400nm.
 18. The nanoparticle of claim 15, wherein the targeting ligandcomprises an RGD peptide, an RVD peptide, or a FROP peptide.
 19. Acomposition comprising at least two blunt-ended siRNA molecules and apharmaceutically acceptable carrier, wherein said siRNA molecules areselected from the group consisting of: 5′-CCUGUUAAUGGGCACACUAGGAAUU-3′(Corresponding to SEQ ID NO: 28) and 5′-CGAGCCGAACCACAACGUCACACAA-3′(SEQ ID NO: 39), and wherein said carrier comprises a dendrimer or ahistidine-lysine polymer.
 20. The composition of claim 19, wherein thepharmaceutically acceptable carrier comprises a histidine-lysinepolymer.
 21. The composition of claim 20, wherein the siRNA moleculesand the histidine-lysine polymer form a nanoparticle whose diameter is100-400 nm.
 22. The composition of claim 19, wherein thepharmaceutically acceptable carrier comprises a dendrimer.
 23. A methodfor treating a human with an HPV infection comprising administering tosaid human a pharmaceutically effective amount of the composition ofclaim
 19. 24. A method for treating a human with an HPV infection and anHIV and/or an HSV infection comprising administering to said human apharmaceutically effective amount of the composition of claim
 22. 25. Amethod for treating a human with an HPV infection and a fungal infectioncomprising administering to said human a pharmaceutically effectiveamount of the composition of claim
 20. 26. A nanoparticle comprising thesiRNA molecules of claim 19, a pharmaceutically acceptable carrier, anda targeting ligand.
 27. The nanoparticle of claim 26, wherein thetargeting ligand comprises an RGD peptide, an RVD peptide, or a FROPpeptide.